Primers were obtained from Sigma-Genosys (Sigma). β-Actin was used as a positive control The sequence of oligonucleotide primers and the product size were as follows: β-Actin (Accession number MMACTBR) 5'-CGTGGGCCGCCCTAGGCACCA, 3'-TTGGCCTTAGGGTTCAGGGGG, 243 bp. Trp53 5' – GGAGTCTTCCAGTGTGATGAT 3'-GGGACAGCCAAGTCTGTTATG 429 bp.

Results

Cultured B6 zygotes developed at a significantly slower rate to the blastocyst stage than did equivalent embryos that developed in the reproductive tract (p < 0.001) (Fig. 1A). Most embryos were morphological blastocysts 89 h after hCG when development occurred in the reproductive tract but less then 20% when development was in vitro. This slower rate of morphological development was accompanied by a slower rate of cell proliferation. Embryos developing in the reproductive tract up to 89 h after hCG administrationpossessedaround twice as many cells as those cultured (p < 0.001) (Fig. 1B). By 113 h after hCG those embryos developing in the reproductive tract had commenced implantation (and were not capable of beingretrieved from the uterus by flushing), while those cultured in vitro were still retarded compared with the fresh 89 h blastocysts (p < 0.01) and still had fewer cells (p < 0.01) than 89 h blastocyst from the uterus (Fig. 1A).

Figure 1

The rate of development of cultured zygotes compared with embryos collected from the reproductive tract at the same time after the administration of ovulatory hCG (h). No embryos could be flushed from the uterus at 113 h since implantation had commenced (not applicable). Numbers shown in brackets are the number of embryos examined. (A) The proportion of embryos that were morphological blastocysts. (B) The total number of cells present in the blastocysts shown in Fig.1A (mean + SEM).

Embryos expressed Trp53 mRNA at all preimplantation developmental stages examined (Fig. 2). TRP53 is generallyconsidered to be constitutively expressed in most cell types, and its cellular concentration is largelyregulated by its rate of degradation. Thus, an understanding of any potentialrole for TRP53 in embryo development is best analyzed at the level of protein expression. TRP53 expression was detected by Western blotting analysis in all preimplantation stages (Fig. 3). Densitometric analysis (Fig. 3) showed that for embryos collected directly from the reproductive tract, expression was at low levels at the pre-compaction stages. In morula stage embryos there was evidence of an increase in expression and expression was higher again in blastocyst stage embryos. For embryos cultured from the zygote stage there were similar levels of TRP53 expression during the pre-compaction stages as observed in embryos collected direct from the reproductive tract. At the morula stage expression was greater and in the blastocyst there was a marked increase in expression in cultured embryos compared with blastocysts collected fresh from the reproductive tract (Fig. 3).

Figure 2

Expression of Trp53 mRNA within preimplantation stage embryos. RTPCR was performed with specific primers to detect β-Actin (positive control) or Trp53. The negative control (No RT) – Trp53 primers but no reverse transcriptase. 1. liver mRNA positive control, 2. oocytes. 3. zygotes. 4. early 2-cell stage. 5. late 2-cell stage, 6. morulae, and 7. blastocyst. Each analysis was performed on groups of 20 embryos and results are representative of 4 replicates.

Figure 3

Expression of TRP53 in embryos at various developmental stages. Densitometric analysis of TRP53 expression as shown in inset. Optical density of p53 band/Lis-1 band *100. The numbers on X-axis represent – Cultured: embryos were collected at the zygote stage and cultured for: (1) 24 h, (2) 48 h, (3) 72 h, (4) 90 h. Fresh: were collected directly from the reproductive tract at the (1) zygote, (2) 2-cell, (3) morula, and (4) blastocyst stages. (5) A positive control for the expression of TRP53 was the analysis of ~1000 T47D breast cancer cells. Inset – Western blotting analysis of 30 embryos at various development stages. After analysis of TRP53 expression membranes were stripped and re-probed for expression of the constitutively expressed Lis-1 protein. The results are representative of 3 replicates. The numbers correspond to those on the graph.

Immunolocalization showed that the increased TRP53 expression in cultured embryos was accompanied by a marked accumulation of TRP53 within the nuclei of some embryonic cells (Fig. 4). It was also shown that production of embryos by IVF followed by culture caused higher levels of TRP53 expression compared with culture of zygotes fertilized in the reproductive tract.

Figure 4

Effect of IVF and culture on TRP53 localization in blastocysts. Localization of TRP53 by indirect immunofluorescence in blastocysts collected from the uterus (Fresh); cultured from the zygote stage (zygotes); or cultured from the zygote stage after production by IVF (IVF). The plate shows images of 2 different representative embryos for each treatment. The experiment was repeated 3 times with a minimum of 8 embryos for each treatment in each replicate.

TRP53 is a transcription factor. It acts to promote transcription of many important proteins, including Bax. The increased TRP53 expression in cultured embryos was apparently transcriptionally active since Western blotting analysis showed that Bax was expressed at high levels in IVF and cultured embryos compared with blastocysts collected direct from the uterus (Fig. 5A). Whole embryo immunolocalization shows that the elevated expression of Bax occurred throughout the cultured blastocysts (Fig. 5B). Bax expression was not observed in cultured Trp53-/-embryos (Fig. 5B). This resultdemonstrates that the enhanced expression of TRP53 in cultured embryos was transcriptionally active.

Figure 5

TRP53 dependent expression of Bax in cultured blastocysts.(A) Western blotting analysis using anti-Bax antibody of blastocysts collected from the uterus on day 4 (Fresh) or of embryos produced by IVF and cultured for 96 h (113 h post hCG). The embryos were extracted as groups of 20, 40 or 80 embryos and then subjected to Western blotting analysis. Approximately 500 F9 cells were also analyzed as positive controls. The blots shown are representative of 3 experiments. (B) The localization of Bax by indirect immunofluorescence (using same antibody as in Western blot analysis) with blastocysts collected from the uterus (Fresh) or those fertilized within the reproductive tract and the cultured for 96 h (Cultured) (113 h post hCG). Controls were subjected to IgG instead of primary antibody (IgG). The cultures were performed with Trp53+/+ and Trp53-/- embryos. Whole section images were deconvoluted and converted to pseudocolor representation of staining intensity. The sections shown are representative of 3 replicate experiments with a minimum of 8 embryos per treatment per replicate.

Embryos are more susceptible to loss of viability in culture if they are cultured individually, compared with those cultured in groups. Figure6 shows that Trp53+/+ zygotes cultured individually had significantly poorer development to the blastocyst stage than did embryos cultured in groups of ten (p < 0.001). This adverse effect of individual culture was significantly (p < 0.01) alleviated in Trp53-/- embryos. The results show that absence of Trp53 did not prevent the retarded development of embryos in vitro in groups, but did prevent the furtherretardation induced by culture at low embryo densities.

Figure 6

The effect of Trp53-/- genotype and the density of zygote culture on the development of zygotes in vitro. The results represent the proportion of zygotes that developed to morphologically normal blastocysts 113 h post hCG. The number of embryos in each treatment is shown in brackets. * shows a significant difference p < 0.01 compared with Trp53-/- embryos.

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Discussion

This study shows that culture of B6 zygotes resulted in their retarded development compared with equivalent age embryos developing in the reproductive tract. Embryos produced Trp53 mRNA throughout the preimplantation stage, confirmingfindings in otherstrains [12]. TRP53 protein was also expressed at relatively low levels during the normal preimplantation phase development. This low level of expression is consistent with the finding that TRP53 is not required for normal development of the embryo in vivo, since Trp53-/- embryos are viable [22]. In blastocysts collected directly from the reproductive tract there is littleobvious accumulation of TRP53 within nuclei and little expression of Bax, a TRP53 transcription product. These observationssuggest a latency of expression and action of TRP53 in the embryo within the reproductive tract. Such latency is to be expected within normal cells [23]. By contrast, the culture of embryos from the zygote stage or their production by IVF (and subsequent culture) resulted in a marked increase in the expression of TRP53 in the post compaction embryos. This increased expression was accompanied by pronouncednuclear accumulation of TRP53. Culture also resulted in increased expression of Bax, and this was TRP53-dependent. Thus, TRP53 expression and nuclear localization in culture embryos was transcriptionally active. This result does not of itself show that Bax is a majoreffector of TRP53 expression in the cultured early embryo. However, the resistance of Bax-/- mouse blastocysts (and partial resistance of Bax+/- blastocysts) to apoptosis induced by the presence of high glucoseconcentrations [24], may suggest that it plays some role. TRP53 induces the expression of many genes and an important research question for the future will be to characterise the TRP53-induced transcriptome in the cultured early embryo in preimplantation stage viability.

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